PHF1 (PHD finger protein 1) is a subunit of the major epigenetic complex, PRC2 (Polycomb repressive complex-2), essential in gene regulation and implicated in cancer and developmental disorders. The methyltransferase activity of PRC2, which is directed at lysine 27 of histone H3, promotes transcriptional repression; however the role of PHF1 in the complex remains unclear. PHF1 contains a unique combination of a Tudor domain fused with a plant homeodomain finger 1 (PHD1) followed by a second PHD2 finger. Our recent studies demonstrate that Tudor recognizes histone H3K36me3, whereas PHD1 binds EZH2, and PHD2 associates with DNA. The molecular mechanisms underlying these novel functions of PHF1 are unknown and will be elucidated in the proposed studies. Detailed knowledge of these mechanisms is of fundamental importance for understanding the physiological and pathogenic activities of PRC2. We hypothesize that binding of the PHD2 finger of PHF1 to DNA stabilizes PHF1 at chromatin, whereas recognition of H3K36me3 by Tudor restricts methyltransferase activity of the PHF1/PRC2 complex. We seek to understand the molecular basis and functional significance of the interactions of PHF1 with chromatin. To determine the mechanism of histone and DNA recognition, the atomic-resolution structures of the PHF1 domains bound to their ligands will be obtained by NMR spectroscopy or X-ray crystallography. Formation of the PHF1-nucleosome complex will be characterized using electrophoretic mobility shift assay (EMSA), NMR and Frster Resonance Energy Transfer (FRET). The mutant proteins impaired in histone or DNA binding activities will be tested in vitro and in vivo by immunoprecipitation, Western blot analysis, ChIP, and histone methyltransferase assays to define the importance of PHF1 in PRC2-dependent transcriptional repression and regulation of histone methylation. These studies will shed light on the role of PHF1 in biological activities of the vital PRC2 complex, allowing us to build a model of signaling by PHF1/PRC2, and will lead to a better understanding of the epigenetic mechanisms of gene transcription.